Analele Universităţii din Oradea, Fascicula:Protecţia Mediului
Vol. XIV, 2009
POSSIBILITIES OF PLANT PRODUCTION FOR BIOREFINERY ON A SOIL
CONTAMINATED WITH HEAVY METALS
Mlinarics E*, Dergez Á*., Blaskó, L.** , Bordás, D* , Zsigrai, Gy.** ,
Kiss, I.*, Szabó, S.**
*Institute for Biotechnology of Bay Zoltán Foundation for Applied Research
H-6726 Szeged, Derkovits fasor 2. [email protected]
**University of Debrecen, CASE, RIC. Karcag Research Institute
H-5300 Karcag Kisújszállási út 166., [email protected]
Abstract
About 17 % of the territory of Hungary is not cultivated and a significant part of this area is not
suitable for agricultural use because of industrial pollution. Green biorefinery technologies allow the
reutilization of hydrocarbon or heavy metal contaminated areas, since agricultural plants and herbs,
which can be sources of valuable products can be produced on polluted sites.
The applicability of biorefinery was tested on a heavy metal polluted soil. The contamination
originated from previous mining activity. Complete by-product utilization from the biomass is aimed
to obtain cosmetic ingredients, pharmaceutical agents and precursors from plants cultivated on
polluted areas. Consequently, we established a technology that ensures the biomass cycle and the
utilization of solar energy bound in plants.
After the survey of the experimental areas (contaminants; microbial florae and endemic vegetation)
88 plant species and varieties were cultivated and tested for potential utilizable components. The
level of possible contaminants in the plants was also monitored and the amounts of different plant
materials were determined (carbohydrates, protein, organic acids, cellulose). Crude plant extracts
were tested as potential sources of biologically effective components (for e.g. antimicrobial
molecules) or as raw materials for lactic acid fermentation. Our results show that biorefinery is a
real possibility for the utilization of polluted areas for industrial purposes. Numerous plants could be
cultivated on contaminated fields without increased levels of contaminants in their tissues, thus they
can be sources of industrially valuable compounds.
Keywords: heavy metal pollution, phytoremediation, plant biomass biorefinery
INTRODUCTION
Biorefinery is a complex technology where biomass can be converted to useful materials
(e.g. fuels, solvents, plastics, cosmetic and pharmaceutical materials and food for humans)
and/or energy carriers in an integrated manner and thereby it can maximize the economic
value of the biomass used while reducing the waste streams produced (Ohara, 2003;
Kaparaju et al., 2009). Use of agricultural lands to produce non-food plants causes social
resistance currently; however, there are some so-called brown field areas (suffering from
industrial contamination), where agricultural output is not feasible due to the high level of
contaminants, such as heavy metal ions. Some plants are able to grow in heavy metal
polluted environment (Murányi and Ködöböcz, 2008; Máthéné and Anton, 2004) and do
not accumulate heavy metals in toxic levels for humans. Murillo et al. (1999) presented that
sorghum and sunflower can easily absorb and translocate heavy metals to plant foliage.
However, phytotoxicity limits the accumulation in plants at safe levels for humans and
animals. On the other hand, various plants, such as lavender contain lots of different
effective compounds, which can be utilized for different industrial purposes, like isolation
of antimicrobial agents (Konstantia et al., 1998; Sato et al., 2007). Moreover, Valcho et al.
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(2008) demonstrated that three essential oil species Marrubium vulgare, Melissa officinalis
and Origanum heracleoticum can be grown as alternative high-value crops on metal
polluted agricultural soils around smelters and provide metal free marketable products.
In 2007 a consortium was generated by the Elgoscar-2000 Ltd., by the Institute for
Biotechnology and the Institute of Logistics and Production Engineering of Bay Zoltán
Foundation for Applied Research, by the Karcag Research Institute of CASE Debrecen
University and by the Biocentrum Ltd for the purpose of execution of a scientific research
program supported by the Hungarian National Office for Research and Technology on the
field of integration of phytoremediation of different polluted fields and of plant biomass
biorefinery. The main goal of the project was to elaborate a new complex technology which
can guarantee the proper treatment and the profitable utilization of polluted fields. For this
purpose biomass production ability and potential availability for industrial utilization of
numerous plant species and varieties were tested in a field experiment and in a pot trial on a
soil extremely polluted by heavy metals.
Some results of the pot experiment are demonstrated in this paper to illustrate our research
work and our screening technologies.
MATERIALS AND METHODS
With the purpose of fulfillment of our scientific program regarding to utilization of fields
polluted by heavy metals a pot experiment (Fig. 1) was carried out at Gyöngyösoroszi in
2008. Great amounts of heavy metal containing slop were arisen at the Mátra Metal Mines
during the ore enrichment processes. Some of this slop was filled into two types of pots.
One type was of 50 dm3 for herbaceous plants and the other type was of 300 dm3 for
arboreal plants. Biomass production capacity, heavy metal tolerance and potential
availability for industrial utilization of 88 different plant species and varieties were tested in
this experiment. The total number of the plots was 428.
Fig. 1 View of the pot experiment
Some chemical properties of the used slop are shown in the Table 1. These analytical data
proved the significant Zn-, Cu-, Cd-, and Pb-pollution of the slop. Ground limestone of 5
m% was mixed with the slop to mitigate the solubility of the heavy metal compounds. The
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used slop did not contain any phosphorus, hence artificial P-fertilization (analogous with
200 kg P2O5 ha-1 dose) was applied in each plot.
Table 1
Some chemical properties of the used slop polluted by heavy metals
pH(H2O)
pH(KCl)
y1
6,63
6,37
10
Zn
Cu
Fe
648
230
258
Zn
Cu
Fe
1715
675
42666
Humic
substances
(%)
1,24
Mg
Na
112
AL-soluble
Ca
(mgkg-1)
21470
1178
43
Co
Ni
Cd
Pb
2,4
2
6,3
356
Co
Ni
Cd
Pb
10
7
7
473
P2O5
K2 O
<2
KCl+EDTA soluble
Mn
Cr
(mgkg-1)
270
4
Total element content
Mn
Cr
(mgkg-1)
964
33
The pot experiment was two-factorial which provided possibilities to examine the tolerance
of different plants (88 species) to heavy metal contaminations and the effects of different
TERRASOL compost doses (analogous with 30 tha-1 and 50 tha-1 doses) on the biomass
production of the tested plant species.
At the beginning of blooming or yield ripening plant samples were taken from the plant
standings of plots to establish the biomass production and to make different chemical
analysis.
Establishment of common plant ingredients
0.2 g plant sample was homogenized in 0.1 N filtered H2SO4 of 5 ml and centrifuged for 20
min (13500 rpm). The supernatant was diluted by H2SO4 to 20-fold and analyzed by
GynkoTek isocratic HPLC arrangement, on CAR-H column and at 30 ºC, and the mobile
phase was 0.01 N filtered H2SO4. Sugars were detected by refractive index detector and the
organic acids were detected by UV detector. Data were integrated by the Chromeleon
program. Cellulose content was determined from 1g plant sample by the nitric acid–ethanol
mixture methodology (Wang Z H, 1995). During the protein assay 0.2 g plant sample was
homogenized in 0.1 N NaOH of 6 ml and incubated in water-bath at 60 °C for 2 hours and
centrifuged for 10 minutes at 13000 rpm. The supernatant was diluted by distilled water to
20-fold. 250 µl taken from the prepared sample was analyzed by the Lowry method (Lowry
et al., 1951). The concentration of the reduced Folin’s reagent is measured by absorbance
with a Unicam Heλios α UV-VIS spectrophotometer at 750 nm.
Heavy metal analysis
Levels of heavy metal contaminants of the plant and soil samples were determined by
Bálint Analitika Ltd. (Hungary) using HP 4500 plus ICP-MS.
Lactic acid fermentation
Shoots of the plants were physically crushed and fractioned by an Angel juice extractor.
The liquid phases were tested as potential medium for lactic acid fermentation.
Lactobacillus delbruckii spp. lactis was applied to convert the sugar content of plant
extracts to lactic acid. The liquid phases were sterilized at 115 °C for 30 minutes then
centrifuged by 5000 rpm for 20 minutes. The inoculum was grown in modified DSM-186
medium and incubated at 37 °C for 72 hours. Inoculum of 20 µl was added to 1 ml
supernatant of the liquid phase and incubated at 37 °C for 48 hours. The initial glucose
content and the amount of produced lactic acid were measured by HPLC.
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RESULTS
Heavy metal pollution tolerance of the tested plant species
On the basis of the experimental data we established that the amount of biomass was
increased by the compost application in the case of a part of the tested plant species and
varieties. Data of Table 2 show some examples of the positive effect of the applied
TERRASOL compost. We must mention that the extremely high compost dose of 50 tha-1
decreased the amount of the green biomass in the case of several plant species.
Under the circumstances of the experiment buckwheat, grain sorhum, energy poplars,
energy willows, hemp, purple coneflower, white mustard and sufflower could reach
relatively high amount of biomass yield.
On the basis of the experimental data originated from the pot trial we established that the
following plant species and varieties could tolerate the unfavorable chemical, hydrological
and microclimatic conditions of the heavy metal polluted trial site: each sweet sorghum
hybrid, castor-oil plant, chicling vetch, amaranth, lozenge, coriander, millet, facelia,
bluebottle, oenothera, evening star, red poppy, Arundo donax, onion, French marigold,
bean, dill, white clover.
From the tested plant species and varieties the following ones could not tolerate the
unfavorable ecological conditions of the experiment: maize, sunflower, pumpkin, lupin,
perennial flax, Canary grass, pine, spruce, oak, wattle, rosemary, milfoil, anise, basil, green
pea, poppy, celery and parsley.
The sensitivity of these plant species to the heavy metal contamination was manifested in
limited germination. Anthocyanine pigmentations were observable on the young seedlings
and their growth was slow. As a consequence of these processed mentioned above, the
sensitive indicator plants died in June and July when additional climatic stress (high air
temperature) was formed out.
Table 2
Effect of compost doses on the green biomass (gm-1) of some plant species grown in
heavy metal polluted soil
(pot experiment at Gyöngyösoroszi, 2008)
Plant species/varieties
grain sorghum (Alföldi 1)
sweet sorghum (Cellu)
grain sorghum (Albita)
grain sorghum (GK Emese)
sweet sorghum (Monori édes)
sweet sorghum (Róna 1)
sudan grass (Gardavan)
grain sorghum (Zádor)
castor-oil plant
sweet sorghum (Sucrosorgo)
hemp
evening star
lozenge
white clover
garlic
French marigold
onion
kitchen sage
Arundo donax
Compost treatments
control
0
0
0
0
0
0
0
0
0
0
0
0
22
46
118
0
0
38
220
749
30 tha-1 compost 50 tha-1 compost
640
320
694
1 032
434
408
416
378
502
706
592
584
370
482
360
306
586
672
196
566
1016
848
286
312
106
156
62
334
200
376
650
664
346
502
384
560
633
426
We observed that the actual biomass production of tolerant plant species covered on a part
of their genetic potential only. By our opinion, the biomass production of these plant
species can be increased by the improvement of the agroecological conditions (water
regime, organic matter content, nutrient supply, etc.) of production sites polluted by heavy
metals.
Analysis of heavy metal contents of the produced biomass
The heavy metal concentrations of the plants grown on contaminated soil were monitored.
After the preparation of plant samples by juicer, 98-100 % of the heavy metals could be
detected in the liquid fraction. We found that heavy metals were concentrated in roots
(Table 3) consequently. The investigated plants did not transport them to the shoots. The
compost treatments had no obvious influences on the heavy metal uptake and distribution.
Table 3
Heavy metal contents of different parts of the selected plant species and varieties
30t/ha
sorghum, Albita
sorghum, Cellu
Cd
50t/ha
30t/ha
Cu
50t/ha
Ni
50t/ha
30t/ha
30t/ha
Pb
50t/ha
30t/ha
Zn
50t/ha
10.40
shoot
0.57
0.19
1.59
0.54
2.09
0.77
0.65
1.49
18.10
root
7.71
1.69
43.20
11.40
8.44
1.51
204.00
193.00
486.00
31.90
shoot
1.46
2.16
1.37
3.42
4.41
0.79
1.40
6.28
30.40
32.60
root
5.62
2.38
26.10
14.50
19.40
2.04
147.00
205.00
94.40
39.10
shoot
0.31
0.31
0.53
2.06
1.31
1.51
0.70
12.80
27.00
21.10
4.78
2.59
17.20
13.60
4.64
2.67
92.20
211.00
85.00
112.00
1.86
0.17
2.67
2.51
2.15
1.93
2.87
1.04
99.80
30.60
6.56
2.59
12.00
49.10
2.72
7.13
384.00
226.00
96.80
110.00
0.44
3.23
0.18
2.02
0.57
9.03
1.05
20.00
2.34
2.06
1.93
2.87
0.97
8.77
1.33
124.00
23.50
186.00
11.40
35.90
12.30
sorghum,GK Emese root
shoot
sorghum, Monori édesroot
shoot
sorghum, Róna 1 root
shoot
0.69
0.11
1.23
0.55
0.46
2.47
1.90
1.00
30.50
root
7.22
1.29
31.60
8.19
2.54
33.70
315.00
32.30
216.00
87.00
balm
shoot
0.97
0.13
74.70
17.20
6.45
1.14
95.20
14.30
136.00
36.10
lavender
shoot
0.23
0.16
37.30
12.40
4.23
1.16
27.90
27.70
78.30
35.50
sorghum, Zádor
Heavy metal recovery was calculated for different sorghum varieties based on biomassyields and the measured heavy metal contents. Similarly to heavy metal distribution, the
varied compost treatment did not cause observable differences in heavy metal recovery
(Fig. 2). However, we would like to highlight that approximately 0.5-1 kgha-1 zinc was
recovered in the “Cellu” and “Monori édes” sweet sorghum varieties. This founding
indicates the possibility of utilization of these varieties for phytoremediational purposes.
Zn
Pb
Ni
Cu
Albita
Cellu
GK
Emese
Monori
édes
Róna 1
50 t ha-1
30 t ha-1
50 t ha-1
30 t ha-1
50 t ha-1
30 t ha-1
50 t ha-1
30 t ha-1
50 t ha-1
30 t ha-1
Cd
50 t ha-1
1000
900
800
700
600
500
400
300
200
100
0
30 t ha-1
heavy metal
uptake g ha -1
Heavy metal uptake
Zádor
Fig. 2. Heavy metal uptake of different sorghum varieties originating from soils
treated by different compost doses (30 and 50 tha-1)
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Analysis of common plant ingredients
Amounts of common plant ingredients, for example cellulose, sugars, organic acids and
proteins were measured in all tested plant species grown on an area extremely polluted by
heavy metals and treated with different compost doses. According to the experimental data,
the varied amount of compost had no effect on the amounts of the monitored plant
ingredients. However, the biomass production was found to be lower on the experimental
site than the usual biomass production on agricultural soils, the proportion of the
compounds were not affected by the presence of heavy metals.
Lactic acid fermentation
The shoots of different Sorghum varieties were ground in an Angel juice extractor, and the
liquid phases of the produced samples were inoculated with Lactobacillus delbruckii spp.
lactis cells. In some cases, the proportion of lactic acid to glucose was higher than one;
consequently, other carbon sources from the plant extract could be used for lactic acid
fermentation by the bacterium. Compost treatment did not affect the effectiveness of lactic
acid production. In some cases the yield of lactic acid was more than 50 g l-1 (Table 4.),
which can be considered as a quite high concentration compared to the literature results
obtained with starch of sorghum seeds (Zhan et.al, 2003.). The high lactic acid
concentration is beneficial for the purification of lactic acid, which might be utilizable to
produce polylactate, a biodegradable plastic( Feerzet Achmad et al. 2009)
Table 4
Lactic acid fermentation from the liquid phase of different sorghum varieties
originated from pots treated with different compost doses (30 and 50 tha-1)
Lactic acid fermentation
Plants
Albita
Cellu
GK Emese
Monori édes
Róna 1
Zádor
30
50
30
50
30
50
30
50
30
50
30
50
t
t
t
t
t
t
t
t
t
t
t
t
glucose g l
-1
ha
-1
ha
-1
ha
-1
ha
-1
ha
-1
ha
-1
ha
-1
ha
-1
ha
-1
ha
-1
ha
-1
ha
40.61
25.29
60.14
20.32
98.54
66.79
53.43
93.85
48.81
182.37
86.82
107.23
-1
produced lactic acid
-1
gl
24.29
8.52
22.37
20.30
54.28
52.25
31.75
53.65
47.09
62.53
60.71
63.85
Volatile compounds
In this study, 20 different, well-known medicinal volatile oils (VOCs) were detected from
the alcoholic extracts of the spice plants. Although, no VOCs were detect from lavender
samples originating from the pots treated with 30 tha-1 compost dose, five different
compounds were found in the samples taken from pots treated with 50 tha-1 compost dose.
The difference in composting also affected the balm content of the plants (Table 5.). The
heavy metal content of the alcoholic plant extracts were found to be low, thus, the further
refining of the extracts for pharmaceuticals or cosmetic base materials seems to be possible.
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Table 5
Volatile oils and heavy metal contents in alcoholic extract of balm and lavender grown
on different compost treated soil.
Volatile oils in balm and lavender
chamfor p-cimene borneol eucaliptol cisze-citral γ-terpinene carvacrol
treatment by
compost Rt=21,885 Rt=19,342 Rt=22,042 Rt=19,513 Rt=22,977 Rt=19,826 Rt=23,66
30 t ha-1
+
+
+
+
balm
50 t ha-1
balm
+
+
+
-1
lavender 30 t ha
-1
+
+
+
+
+
lavender 50 t ha
plants
Heavy metal content in the plant extracts
Zn
Cu
Pb
Cd
Ni
0,00
7,84
7,84
0,00
0,00
0,00
0,00
0,00
7,92
0,00
0,00
1,98
1,98
0,00
0,00
0,00
0,00
0,00
0,00
0,00
Antimicrobial effects
The sterile filtered liquid fraction of some sorghum varieties, like Albita and Zádor showed
antiviral activity in the 8-fold dilution. The 16-fold dilutions were too diluted to exert their
effect on VSV and the concentrated ones were toxic to Hep2 tissue.
The sterile filtered liquid fractions of plant samples were tested for containing potential
biologically effective components, such as antimicrobial compounds. The antibacterial
activity was tested against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus
aureus. The tested plants did not have any antibacterial effect against P. aeruginosa and E.
coli, however, some sorghum varieties, like Monori édes and Cellu, had antibacterial
activity against S. aureus. The inhibition zone had the same size as 30 µg ml-1
chloramphenicol (Fig. 3).
Inhibiton of
Staphylococcus aureus
by Chloramphenicol 30µg
Inhibiton of
Staphylococcus aureus
by extractum of sweet Sorghum
Fig. 3. Antibacterial effect of sweet Sorghum
Some of the samples demonstrated antifungal activity against the Paecilomyces
variotii only, however, this fungi was sensitive for all investigated plant extracts.
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CONCLUSION
On the basis of the experimental data we established that some plant species can adapt to
the unfavorable ecological conditions of heavy metal polluted soils. The amount of biomass
of these plant species was increased by the compost application significantly.
According to the results, numerous plant species were able to synthesize some biologically
active compounds, like antimicrobials, without the accumulation high level of heavy metal
contaminants. These results suggest that plants cultivated on industrial, heavy metal
contaminated areas can serve as valuable sources of base materials for biorefinery.
As a next step, the development of a complex purification method of the active compounds
is necessary, considering the presence of heavy metal contamination.
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Acknowledgements
This work was supported by the grants NKFP_07_4-BIOFINOM.
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